Asthma is characterized by variable, reversible airflow obstruction, and airway hyperresponsiveness (AHR), associated with an infiltration of the bronchial mucosa with activated T-lymphocytes (T-cells), and eosinophils. These cells, along with resident airway mast cells, secrete a variety of cytokines and mediators that play a fundamental role in the pathogenesis of the disease. CD4+ Th2 cells, through the release of specific cytokines (IL-4, IL-5, IL-9, and IL-13), are thought to orchestrate the disease process (1,2). In particular, the Th2 cytokines IL-4, and IL-13, are considered pivotal to the development and maintenance of airway inflammation and airway hyperresponsiveness.
A number of in vivo studies also support the pivotal role of IL-4 and IL-13 in the pathogenesis of asthma. Using animals deficient in either cytokine, or reagents that neutralize either IL-4 or IL-13 function, an important role of these cytokines is observed in regulating the primary and secondary immune response leading to airway inflammation and airway hyperresponsiveness (3, 4). Cumulatively, these data suggest that IL-4 and IL-13 may play both overlapping and independent roles in the allergic airways response, and that targeting both cytokines could have significant added benefit to targeting either cytokine alone.
Antagonists of IL-4 have been reported in the literature. Mutants of IL-4 that function as antagonists include the IL-4 antagonist mutein IL-4/Y124D (Kruse, N., Tony, H. P., Sebald, W., Conversion of human interleukin-4 into a high affinity antagonist by a single amino acid replacement, Embo J. 11:3237-44, 1992) and a double mutein IL-4[R121D/Y124D] (Tony, H., et al., Design of Human Interleukin-4 Antagonists in Inhibiting Interleukin-4-dependent and Interleukin-13-dependent responses in T-cells and B-cells with high efficiency, Eur. J. Biochem. 225:659-664 (1994)). The single mutein is a substitution of tyrosine by aspartic acid at position 124 in the D-helix. The double mutein is a substitution of Arginine by Aspartic Acid at position 121, and of tyrosine by aspartic acid at position 124 in the D-helix. Variations in this section of the D helix positively correlate with changes in interactions at the second binding region.
Mutant variants of IL-4 demonstrating agonism or antagonism of wild-type IL-4 may be useful for treating conditions associated with one of the pleiotropic effects of IL-4. For instance, antagonists of IL-4 would be useful in treating conditions exacerbated by IL-4 production such as asthma, allergy, or other inflammatory response-related conditions. Agonists of IL-4 may be useful for treating conditions wherein the presence of IL-4 is associated with the amelioration or attenuation of a disease, for example, an autoimmune disease such as Rheumatoid Arthritis, Multiple Sclerosis, Insulin-dependent Diabetes Mellitus, etc. These autoimmune diseases are characterized by a polarization in production of the T helper cell populations, types 1 and 2 (Th1, Th2). Naive CD4+ T cells differentiate into Th1 or Th2 subsets, depending on the cytokine present during stimulation. An IL-4 agonist would ideally shift production to the T-helper cell desired, i.e., towards Th2, thereby having a therapeutic effect.
PCT/US93/03613 discloses an IL-4 variant having a Phe-Leu or Tyr-Leu sequence in a alpha-helical domain and a negatively-charged amino acid within two amino acids immediately upstream or downstream from the Phe-Leu or Tyr-Leu sequence, the variant having an increased affinity for the IL-4 receptor by virtue of a neutral amino acid substituted for the negatively-charged amino acid. It also discloses that the specific substitution of Trp-Leu or Phe-Leu within an a-helix of IL-4 within 2-residues of a negatively charged residue results in improved affinity. The variant is an IL-4 fusion protein (with diphtheria toxin).
A recombinant mutein protein (IL-4RA) derived from human IL-4 mutated in two positions of its amino acid sequence was previously reported in U.S. Pat. Nos. 6,028,176 and 6,313,272. IL-4RA binds with high affinity to the human IL-4 receptor alpha chain, an important functional signaling component of both the IL-4 and IL-13 receptor complexes. This mutein has no agonist activity, and acts as a potent competitive IL-4 and IL-13 receptor antagonist in vitro (See U.S. Pat. Nos. 6,028,176 and 6,313,272). A significant drawback to the use of IL-4RA is its relatively short half life in vivo (approximately 3-6 hrs). Pharmacokinetic/pharmacodynamic modeling of IL-4RA in the primate asthma model indicates that the effective average steady state concentration for optimal therapeutic effect is approximately 60 ng/ml.
One approach to overcoming the short half life is frequent administration of the IL-4RA mutein to a patient, however frequent administration (usually by injection or tracheal intubation) creates very significant barriers to patient acceptance of the therapy and therapeutic administration in a clinic.